Impotence, the inability to obtain or maintain a penile erection sufficient for sexual intercourse, afflicts more than 12 million men in the USA. It is associated with aging and occurs in 25% of men aged 65, and 55% of men aged 75, irrespective of the fact that the libido of the majority of these patients is relatively unaffected. Annually it results in more than 400,000 outpatient visits and 30,000 hospital admissions in the U.S. Surgical implantation of penile prosthesis increased from 19,000 in 1989 to 32,000 in 1989. Accordingly, the costs of treating impotence in 1989 is conservatively estimated at 250 million dollars. In human terms, although impotence is not usually a life-threatening situation, its consequences for the patient and his partner are psychologically very serious.
Contrary to earlier assumptions in the literature, as much as 90% of impotence is due to organic and not to psychogenic causes. Despite the fact that aging is a predisposing factor, organic impotence may be present as early as puberty in some patients. Vasculogenic and neurogenic alterations leading to penile erectile dysfunction are at the root of the majority of the organic impotence syndromes, since male hormone disturbances and other possible physical causes of loss of the libido play a minor role in these problems.
Vascular disease of many causes will eventually lead to impaired penile erection. Thus, atherosclerosis, hypertension, diabetes, heavy smoking and alcoholism are all recognized risk factors for erectile dysfunction. Diabetics as a group are the most prone to vasculogenic impotence with more than 50% incidence in a population of about 2.5 million in the USA.
This invention is based on the discovery that effective ameliorative treatment can be based on inducing the penile tissue specific expression of nitric oxide synthase, the enzyme which synthesizes the compound nitric oxide (NO), which in turn functions as a mediator of penile erection.
The physiology of normal erection can be divided into three distinct processes acting in concert: (a) increased arterial inflow; (b) decreased venous outflow; (c) active cavernosal smooth muscle relaxation. The latter appears to be the key event, but the penile blood vessel hemodynamics is also mediated by the smooth muscle of the arterial tree. Accordingly, active smooth muscle relaxation in the penile artery and sinusoids is considered to be the pivotal step in generating a normal erection. Abnormalities in penile smooth muscle function may be the critical site in erectile dysfunction.
In a normal erection the stimulation is transmitted to the penis through the nervi erigentes, the pelvic autonomic nerve fibers. Neurotransmitters are released from three systems:
(a) norepinephrine from the sympathetic adrenergic fibers; PA1 (b) acetylcholine from the parasympathetic cholinergic fibers; and PA1 (c) a substance from the nonadrenergic-noncholinergic (NANC) fibers. The NANC neurotransmitter has been shown to be nitric oxide (NO) and to act upon the smooth muscle to cause relaxation.
The smooth muscle relaxation of the trabeculae surrounding the lacunar spaces of the corpora cavernosa has three important functions: (a) reduction of the normally high resting (flaccid) resistance to arterial flow, thus increasing this flow through the helicine arteries into the endothelium-lined lacunar spaces; (b) regulation of blood storage into the penis, allowing penile engorgement; and (c) transmission of approximately 80% of systolic blood pressure into the cavernosal space. The latter will compress the draining venules that run in parallel between the expanding smooth muscle and the tough inelastic tunica albuginea, resulting in venous outflow restriction. Detumescence occurs by a reversal of this process, that is, an increase of the tone of the smooth muscle in both compartments leading to reduction of arterial inflow and the size of the lacunar spaces, followed by venous runoff.
NO and the endothelium-derived relaxing factor (EDRF) appear to play multiple roles in different biological processes. NO is considered to be responsible for the vasodilator activity of EDRF which is released from the endothelial lining of blood vessels and induces different effects in hypoxia, vascular disease, septic shock, and inflammation. EDRF plays a significant physiological role in the maintenance of vascular tone by inducing locally the relaxation of the smooth muscle cells. Our work has shown that in the penile corpora cavernosa, NO may be the NANC neurotransmitter in the penis and the main compound responsible for erection.
In the case of the penis, we have previously demonstrated by electric field stimulation (EFS), pharmacological treatments and the use of specific NOS inhibitors, that NO is the main mediator of penile erection in the human, dog, rat, and rabbit. A number of other laboratories have confirmed and extended these findings, by applying essentially two approaches: a) relaxation of corpora cavernosa strips in organ bath; b) erectile response in animal models. The latter procedure has been employed by us in a rat model to define the main object of this invention.
One of the sites of NO release in the penis appears to be at the non-adrenergic non-cholinergic (NANC) nerve terminals of the corpora cavernosa, from where it then binds to guanyl cyclase in adjacent cells and stimulates the formation of cGMP mainly in the smooth muscle target tissue. This cGMP synthesis in turn results in a decrease in intercelluar Ca2+ and subsequent smooth muscle relaxation and penile vasodilation.
It is known in the art that Nitric Oxide Synthase (NOS) is the enzyme catalyzing the formation of NO in endothelial cells, macrophages, brain, liver and several other cell types and tissues. There are two types of NOS: constitutive NOS (cNOS), whose levels do not appear to change upon different experimental conditions; and inducible NOS (iNOS), whose synthesis can be stimulated by bacterial toxins and certain growth factors. In general, cNOS is present in brain (one isoform of which is known as neural NOS, or nNOS), and in endothelial cells, while iNOS is contained in macrophages, lung, liver, smooth muscle cells from large arteries and also in endothelial cells.
cNOS and iNOS isolated from different tissues show the existence of several isoforms within each group with specific cofactor requirements, mRNA sizes, and immunological properties. (The different type designations are shown below in parenthesis.) Within the cNOS group, characterized by their Ca.sup.2+ dependence, there are three different cytosolic isozymes: (Ia), present in the brain, cerebellum, neuroblastoma cells; (Ib), present in endothelial cells; and (Ic), present in neutrophils. The first two are calmodulin dependent, and the third is calmodulin independent. Ib does not have BH.sup.4 and FAD as cofactors, and Ia is the only one using FMN additionally as cofactor. There is also a particulate Ca.sup.2+ /calmodulin dependent isoform that makes up over 90% of endothelial cell cNOS.
Within the iNOS group (all Ca.sup.2+ /calmodulin independent with unknown regulators), a soluble type is present in the macrophages (II), hepatocytes, Kupfer cells, fibroblasts, endothelial cells, lung and liver, and has all the requirements of isoform Ia. A different particulate type (IV) is present in the macrophages and is only NADPH dependent. Recently, a new nomenclature has been proposed based on three NOS types: "I" or neuronal cNOS (nNOS) which is the brain cNOS; "II" or endothelial NOS (eNOS); and "III" or inducible NOS (iNOS).
Further evidence for the significant difference between cNOS, nNOS and iNOS is evident from the difference in their respective kinetics and substrate/cofactors requirements. L-arginine and NADPH are the common substrate and cofactor respectively. As noted above, the cNOS/nNOS and iNOS isozymes can be distinguished in that cNOS/nNOS is Ca.sup.2+ /calmodulin dependent, while iNOS is stimulated by tetrahydrobiopterin. NOS activity is inhibited by a series of competitive inhibitors such as N.sup.G -nitro-L-arginine and N.sup.G -methyl-L-arginine, or by NO chelators, such as hemoglobin. (3-H)citrulline synthesis is increased in certain cells by N-methyl-D-aspartate (NMDA) and glutamate. L-nitroarginine is 1000-fold more potent inhibitor of the cNOS than of the iNOS. Hydroxy-arginine and arginine dipeptides are also NOS substrates. Aminoguanidine appears to be a preferential inhibitor for iNOS, but its specificity varies with the cell type.
In addition, NOS mRNA (and resulting cDNA) are proving to be highly species and tissue specific. For example, the DNA sequence (including introns and exons) of the gene for rat brain cNOS (nNOS) has been cloned. Its mRNA is expressed as a 10.5 kb polynucleotide species. But the same mRNA species is not found in rat kidney, liver, skeletal muscle or heart tissue. Besides rat cerebellum, cNOS has been purified from rat polymorphonuclear neutrophiles. The cNOS cDNA has also been cloned from bovine and human endothelium. In the latter case, the corresponding cNOS mRNA is 4 kb in length, and is encoded by a gene different from that expressed in the brain. Further, iNOS has been purified from LPS-stimulated rat and mouse macrophages, rat vascular smooth muscle, human hepatocytes and condrocytes. Induction of NOS is triggered in vivo by injection of lipopolysaccharide from E. Coli (LPS), and in vitro by incubation of cells or tissue strips with LPS, interleukin .beta., and tumor necrosis factor (TNF-.alpha.) interferon. The induction is protein synthesis-dependent and blocked with dexamethasone or other glucocorticoids, and with TGF-.beta..
The presence of NOS in the human, rabbit, and rat penis tissue homogenate has been shown by us by following the conversion of (3-H) L-arginine into (3-H) L-citrulline in the cytosol fraction, and others have detected the nNOS isoform in the nerve terminals of the penis by histochemistry and immunocytochemistry. However, no characterization has yet been made of the main penile NOS isozyme responsible for NO synthesis during sexual stimulus. In addition, recent gene knock-out experiments failed to affect the reproductive behavior of transgenic mice when nNOS was silenced. Our own current work indicates the presence of distinctive penile NOS isozymes different from those in other tissues. Other non-NOS dependent pathways may be present in the penis and they are supposed to cooperate during penile erection with the NO cascade, or become predominant after a long-term impairment or silencing of the penile NOS. These putative physiological ancillary relaxants of the penile smooth muscle include vasoactive intestinal polypeptide (VIP), calcitonin gene related polypeptide (CGRP), prostaglandins, etc.
That NOS decrease or inactivation may be associated with certain forms of erectile dysfunction has been shown by our recent work on aged intact rats, diabetic BB rats, and castrated rats. In both intact senescent rats and castrated rats, the levels of erectile response to EFS and of penile NOS can be restored to normal values by androgen administration. EFS itself modulates penile NOS activity, and it does this in a differential form between intact and castrated rats.
However, no treatment based on the manipulation of endogenous NO synthesis or of NOS activity or expression has been proposed in the literature. The current pharmacotherapy of erectile dysfunction is based exclusively on the topical application or the direct intermittent self-injection into the penile corpora cavernosa of mixes of vasoactive compounds, including nitrodonors, immediately prior to sexual intercourse, or surgical treatments based on prosthesis implantation or arterial/venous operations. For example, U.S. Pat. Nos. 4,931,445 (Goldstein et al.), 5,336,678 (Cavallini), and 5,278,192 (Fung et al.), teach methods of treating impotence through administering the drugs etoparidone, Minoxidil, or isobutyl or isoamyl nitrite, respectively. Bredt et al. in U.S. Pat. No. 5,268,465 have characterized a rat brain cDNA encoding a calmodulin-dependent NOS molecule of specific sequences, but does not suggest or teach treatment of erectile dysfunction therewith. This appears to be a cNOS or nNOS. Stuehr et al. in U.S. Pat. No. 5,132,407 teach a three-component calmodulin-independent cNOS flavoprotein purified from mouse macrophages, but does not teach treatment of erectile dysfunction.
Voss et al. in U.S. Pat. No. 4,801,587 teaches use of DMSO as an absorption agent to introduce papaverine, a compound known for treatment of human impotence. El-Rashidy in U.S. Pat. No. 5,256,625 teaches the use of hydroxy propyl-.beta.-cyclodextrin as an absorption enhancer for papaverine.
The subject of the current invention, the penile iNOS isoform, has never been detected either at the enzymatic or protein levels, at the mRNA levels, nor in penile tissue sections by immunocytochemical procedures. Other than our own work on rat penile smooth muscle cells (RPSMC) described herein there are no in vitro reported studies on iNOS detection in cultures of penile cells. It is presently unknown whether penile iNOS has any physiological role, and there are no publications suggesting that it could be applied for the therapy of erectile dysfunction. In fact, vascular iNOS in general, when induced, may have a deleterious effect on blood pressure. It is assumed to participate in septic shock, without apparently acting on the normal maintenance of blood vessel tone. In addition, the induction of iNOS to improve penile erection has not probably been considered before because of the risk of systemic hypotension or uncontrollable priapism. To our knowledge, no publications on the continuous delivery of compounds into the penis are available.
Accordingly, there is a need in this field to provide an improved method of treatment of erectile dysfunction by inducing endogenous production of iNOS in penile tissue or by introduction of exogenous iNOS to penile tissue.